Targeted manipulation of gene expression is a fundamental concern in crop biotechnology. A variety of molecular methods has been developed to manipulate gene expression. However, targeted gene inactivation is still practically difficult in most crop species. RNA interference (RNAi), which is frequently used for targeted gene silencing, often suffers from off-target effects and unstable gene suppression. Engineered nuclease-based tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been developed to induce site-specific genome modifications. These approaches facilitate precise genetic modifications but require much time and labor for extensive screening.
It has recently been reported that the activities of dimeric transcription factors are efficiently suppressed by genome-encoded siPEPs that competitively form nonfunctional heterodimers in plants (Seo et al., Trends Plant Sci. 16, 541-549, 2011). The LITTLE ZIPPER (ZPR) proteins consisting of 67-105 residues contain leucine zipper motifs and interact with class III homeodomain-leucine zipper (HD-ZIP III) transcription factors. However, they lack protein domains required for DNA binding and transcriptional activation. As a result, the ZPR proteins attenuate the HD-ZIPIII transcription factor activities by reducing DNA binding affinity and transcriptional regulation activity. Similarly, the MINI FINGER (MIF) proteins interfere with zinc finger-homeodomain (ZHD) transcription factors, which function in multiple hormone signalings and floral development, by inhibiting nuclear import and DNA binding of the target transcription factors (Hong et al., J. Biol. Chem. 286, 1659-1668, 2011).
Notably, the siPEPs are also produced by alternative splicing of transcription factor genes. The Arabidopsis INDETERMINATE DOMAIN 14 (IDD14) gene undergoes alternative splicing, producing two spliced isoforms, designated IDD14α and IDD14β. The IDD14β form has disrupted DNA-binding domain but is able to form heterodimers with the IDD14α □ form (Seo et al., Nat. Commun. 2, 303, 2011b), further extending the repertoire of the siPEP in plants.
In animals, a variety of cancers are caused by constitutive expression of transcription factor genes. It has been shown that synthetic peptides, when injected into animal tissues, efficiently repress the activities of oncogenic transcription factors (Polo et al., Nat. Med. 10, 1329-1335, 2004). In addition, dynamic dimer formation of transcription factors, such as STAT3 (signal transducers and activators of transcription 3), c-Myc, Max, c-Jun, and c-Fos, is disturbed by peptidomimetics, small protein-like molecules that structurally mimic protein domains required for dimerization. The small molecules suppress transcription factor activities by inhibiting either protein-protein interactions or DNA binding, similar to what have been observed with genomic siPEPs in plants.
Inventors of the present invention explored the biotechnological relevance of engineered transcription factor proteins, which designated artificial siPEPs (a-siPEPs) because of their structural mimicry to genomic siPEPs in plants. Gene sequences encoding potential a-siPEPs of SOC1 and AG MADS box transcription factors, which function in flowering induction and floral organogenesis, respectively (Komeda, Annu. Rev. Plant Biol. 55, 521-535, 2004), and the LHY MYB transcription factor involved in circadian clock control (Schaffer et al., Cell, 93, 1219-1229, 1998) were transformed into Arabidopsis and Brachypodium. Phenotypic comparison and biochemical and molecular analyses revealed that the a-siPEPs inhibit nuclear import and DNA binding by forming nonfunctional dimers in both plant species, supporting that the a-siPEP tool could be employed to inactivate specific transcription factors in crop plants.
Meanwhile, in Korean Patent Application Publication No. 2012-0017913, “Inhibitor of transcription factor having fusion protein which contains DNA binding domain and protein transport domain of transcription factor and preparation method thereof” is disclosed. Further, in Korean Patent Application Publication No. 2007-0076918, “Plants enhanced by the development of wide-leafed, late-flowering and environmental stress-resistant by transforming the transcription factor gene AtMYB44” is disclosed. However, the method for targeted inactivation of transcription factor using an artificial small interfering peptide and uses thereof that are described in the present invention have never been disclosed in any of those literatures.